The effects of TSH and the activation of the cyclic AMP (cAMP) and Ca2+-phosphatidylinositol (Ca2+-PI) cascades on the activity and expression of the selenoenzyme thyroidal type-I iodothyronine deiodinase (ID-I) have been studied using human thyrocytes grown in primary culture. Stimulation of ID-I activity and expression was obtained with TSH and an analogue of cAMP, 8-bromo-cAMP. In the presence or absence of TSH, the addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) together with the calcium ionophore A23187, caused a decrease in ID-I activity; a decrease in ID-I expression was also observed as assessed by cell labelling with [75 Se]selenite. PMA alone had no effect on ID-I activity in the presence or absence of TSH. A23187 alone produced a small but significant reduction in ID-I activity, but only in TSH-stimulated cells. These data provide evidence that the expression of thyroidal ID-I is negatively regulated by the Ca2+-PI cascade, and positively regulated by the cAMP cascade.